Rheumatology 2001;40:623±630

Osteoprotegerin and receptor activator of nuclear factor kappaB RANKL) regulate formation by cells in the human rheumatoid arthritic joint

D.R.Haynes,T.N.Crotti,M.Loric,G.I.Bain1,G.J.Atkins andD.M.Findlay1 Department of Pathology and 1Department of Orthopaedics and Trauma, The University of Adelaide and The Royal Adelaide Hospital, Adelaide 5000, SA, Australia

Abstract Objective. This study investigated the involvement of the recently identi®ed regulators of osteoclast formation RANKL wreceptor activator of nuclear factor kappaB RANK) ligand, osteoclast differentiation factor, TRANCE, ligandx and its natural inhibitor, osteoprotegerin OPG), in the of RA). Methods. mRNA was extracted from cells isolated from the pannus and synovial membrane regions of joints of 11 RA patients. Semiquantitative reverse transcription±polymerase chain reaction was carried out, and the isolated cells were also cultured to determine their ability to form . Results. mRNAs encoding RANKL, RANK, OPG and -colony stimulating factor were expressed by cells isolated from RA joints. In addition, mRNA encoding for tumour necrosis factor -inducing ligand and the osteoclast markers tartrate-resistant acid phosphatase and calcitonin receptor were also often expressed. Osteoclasts capable of forming resorption lacunae were generated from cells in the RA joints. At 50 nguml, recombinant OPG completely inhibited the resorptive activity of these cells. There was a signi®cant correlation between the ratio of RANKL mRNA to OPG mRNA and the number of resorption pits produced P = 0.028). Conclusion. These data suggest that RANKL is an essential factor for osteoclast formation by cells in the rheumatic joint and that OPG may prevent the bone erosion seen in RA joints.

KEY WORDS: Bone, Rheumatoid arthritis, Joint damage, , Osteoclasts, Osteoprotegerin.

Progressive joint destruction is a hallmark of rheuma- differentiation factor, TRANCE and osteoprotegerin toid arthritis RA). The in¯ammatory changes in RA ligand) and its receptor, receptor activator of nuclear are associated with the breakdown of both soft kappaB RANK) have been shown recently to and bone in the rheumatoid joint. The bone erosion can be key factors stimulating osteoclast formation w3, 4x. be localized to the in¯amed joint w1x as well as being It has been shown that the binding of RANKL to generalized, secondary often being associ- RANK on the surface of osteoclast precursors promotes ated with RA w2x. Despite the widespread occurrence the differentiation of these cells to mature osteoclasts. of RA, we still have an incomplete understanding of It is now clear that, together with macrophage-colony the processes of this chronic systemic disease. Most stimulating factor M-CSF), RANKL is required for studies have investigated the in¯ammation that occurs in osteoclast formation. The soluble tumour necrosis factor the soft tissues; however, recent advances in our under- TNF) receptor-like molecule osteoprotegerin OPG) standing of bone metabolism allow us now to better is a natural inhibitor of RANKL w5x. OPG binds investigate the mechanisms of bone loss in RA. to RANKL and prevents its ligation to RANK. The A cell surface molecule, receptor activator of nuclear importance of these molecules in regulating bone meta- factor kappaB ligand RANKL; also called osteoclast bolism is demonstrated by transgenic and knock- out studies in mice w6x. The relative levels of RANKL and OPG are likely to be important in determining Submitted 18 May 2000; revised version accepted 11 December 2000. whether osteoclasts will form w7x. As these factors con- Correspondence to: D. R. Haynes. trol physiological osteoclast formation, it is reasonable

623 ß 2001 British Society for Rheumatology 624 D. R. Haynes et al. to propose that they may also be key regulators of surgery by the surgeon co-author GIB); the pannus pathological , such as in RA. tissue was considered to be that part of the synovial It has been reported previously that, under certain membrane that had in®ltrated the bone and cartilage. conditions, human osteoclasts can be derived from The tissue classi®ed as synovial membrane was capsular cells present in or near the tissues of arthritic joints synovial tissue separate from bone±cartilage in®ltrate. w8, 9x. More recently, it has been shown that RANKL Histology was carried out routinely on samples of these is expressed within the rheumatoid joint and that tissues and examples are shown in Fig. 1. Generally, the synoviocytes and activated T cells are implicated in samples were similar and contained large numbers of its production w10±13x. Moreover, in a mouse model in®ltrating mononuclear cells. The only major difference of RA, administration of OPG prevented the bony was that the pannus tissues did not have a layer of cells that often accompany joint in¯ammation in lining the surface of the synovial membrane. On RA w12x. In the present work, we sought to test the removal, the tissue samples were placed immediately concept that the production of RANKL by cells within into Hanks' balanced salt solution HBSS; Gibco BRL, the human RA pannus and synovial membrane leads to Life Technologies, Melbourne, Australia) then digested osteoclastic bone resorption. Cells isolated from the at 378C in calcium- and magnesium-free HBSS solution synovial membrane and at the bone±pannus interface Gibco BRL, Life Technologies) containing 1 mguml were used in this study. We found a positive associa- tion between RANKL expression and bone resorption in vitro, and that resorption was completely prevented by exogenous OPG.

Materials and methods Chemicals Human recombinant RANKL and OPG were gifts from Amgen Thousand Oaks, CA, USA).

Patient tissue samples and cell isolation Tissue samples were taken at surgery from patients who had been diagnosed as suffering from RA, which they had had for 6±25 yr. All patients showed advanced erosion of the bone on X-ray, and had disease that was active but not burnt out, as seen in the late stages of the disease. Details of the samples taken from the patients and of the medication at the time of surgery are shown in Table 1. The tissue samples were classi®ed as corresponding to either the pannus region adjacent to the bone where erosion was occurring, or to the synovial membrane not adjacent to the bone. In all cases the classi®cation of the tissue was made at the time of

TABLE 1. Details of samples from patient tissues

Disease Patient duration sample Age Sex yr) Medications Site Region

1 58 M 17 Gold, prednisolone Wrist Pannus 2 71 F 19 NSAID Knee Pannus 3a 37 M 15 NSAID Elbow Pannus 3b Elbow Membrane 4 48 F 20 Gold Wrist Pannus 5 52 F 30 Methotrexate Wrist Membrane 6 44 F 6 Herbal therapies Wrist Pannus only FIG. 1. a) Typical section of synovial membrane. This 7a 51 M 17 Methotrexate Foot Membrane consisted of a membrane lining layer of cells and a sublining 7b Foot Pannus layer of connective tissue of variable thickness in®ltrated 8 57 F 25 NSAID Wrist Pannus by a large number of mononuclear cells. b) Pannus tissue 9 52 F 22 Methotrexate Wrist Membrane was very similar but lacked the layer of cells that lined 10 50 F 9 Methotrexate Elbow Membrane 11 57 F 15 Gold, prednisolone Wrist Pannus the membrane of the synovium. Both sections were formalin- ®xed, paraf®n-embedded and stained with haematoxylin. NSAID, non-steroidal anti-in¯ammatory drug. Magni®cation 3100. Factors regulating bone loss in RA joints 625 collagenase Sigma, Castle Hill, Australia) and 1 mguml Preparation of total RNA and RT-PCR analysis dispase Sigma). After 60 min, 0.5 mguml trypsin in The total cell population isolated from pannus or HBSS solution Sigma) was added, and the tissue was synovial membrane, as described above, was lysed by incubated for a further 30 min. Cells were separated the addition of Trizol reagent Life Technologies, from undigested connective tissue with a 70-mm cell Gaithersburg, MD, USA) and total RNA was prepared sieve Falcon, Becton Dickinson Labware, Bedford, according to the manufacturer's instructions. cDNA MA, USA) and the cell suspension was washed once in was synthesized using an AMV RT cDNA HBSS. Cells were suspended in RPMI 1640 medium at Promega, Madison, WI, USA). cDNA was ampli®ed 6 a concentration of 1 3 10 cellsuml. by PCR in a thermal cycler Eppendorf, Hamburg, Cells were isolated from a total of 11 patients with Germany). Each ampli®cation mixture contained 1 ml RA, and tissue was taken from two separate sites in two of the cDNA sample or water control, 0.2 mM dNTPs patients. The yield of cells varied depending on the size and 1 U of Platinum Taq DNA polymerase Life and the cell density of the tissue sample. An average of Technologies), 100 ng each of 59 and 39 primers, 1.5 mM 5 3 105 cells from each milligram of wet weight of tissue MgCl2, 2 ml103 reaction buffer, and sterile diethyl was obtained. mRNA was extracted from 5 3 105 cells pyrocarbonate H2O. Twenty-two cycles of PCR were for analysis by reverse transcription±polymerase chain performed for glyceraldehyde-3-phosphate dehydro- reaction RT-PCR). Suf®cient cells for the studies of genase GAPDH) and 30±34 cycles for the other osteoclast generation, as described below, were obtained primer pairs. Primer sequences and predicted PCR from eight of the patients. product sizes have been published previously w15, 16x. Ampli®cation products were resolved by electrophoresis Isolation of human bone-derived cells on a 2% wuv agarose gel and post-stained with SYBR Human bone cells were derived as outgrowths from Gold catalogue no. S-11494; Molecular Probes, trabecular bone fragments obtained from patients Eugene, OR, USA). The intensity of the PCR products undergoing primary hip replacement, as described was quanti®ed using a Molecular Imager Fx ¯uorescent previously w14x. scanner and Quant-1 software Bio-Rad, Hercules, CA, USA). Preliminary experiments were performed to Culture of osteoclasts and osteoclast precursors ensure that the number of PCR cycles was within the Cells isolated from the rheumatoid tissues were tested exponential phase of the ampli®cation curve. This for osteoclast formation anduor activity, as described allowed semiquantitative comparisons to be made previously in detail in studies in which the differentia- between the levels of expression of the various RNA tion of monocytes to osteoclasts was promoted by species in the samples, as described previously w15, 16x. coculture with rodent osteoblastic cells as stromal cells w15x. Brie¯y, where human bone cells were used as a Results stromal population, 13-mm diameter sterile glass cover- slips or 3.0 3 0.1-mm thick discs of dentine were seeded Expression of regulators of osteoclast with human bone-derived cells 24 h before addition of formation in rheumatoid tissues 'RT-PCR) cells isolated from the rheumatoid tissues. Rheumatoid Figure 2 shows PCR products corresponding to mRNA cells w4 3 105 coverslips) or 2 3 105 dentine)x were for RANKL, OPG and M-CSF, as determined by added and after 1 h the non-adherent cells were RT-PCR, in cells digested from rheumatoid tissues removed by washing. The individual coverslips and sampled from 11 patients. PCR products corresponding pairs of dentine slices were placed in 16-mm diameter 2 8 to each of the mRNA species could be ampli®ed wells with 1 ml of aMEM medium containing 10 M 2 8 from nearly all tissue samples. We also observed that 1a,25 OH)2D3 vitamin D3), 10 M dexamethasone , a TNF-a-related molecule that has been shown Fauldings, Adelaide, Australia) and 25 nguml recombi- to bind to and antagonize the inhibitory actions of OPG nant human M-CSF a kind gift from the Genetics w17, 18x, was expressed in all the samples tested. mRNA Institute, Cambridge, MA, USA). Medium was replen- encoding for the osteoclast markers TRAP and calci- ished every 3 days throughout the experiment and all tonin receptor CTR) were also expressed in many of experiments were carried out in duplicate for each the rheumatoid tissues. TRAP was expressed in all but rheumatoid sample. two samples. In contrast, CTR was expressed in all the pannus tissue samples but only one of the synovial Tartrate-resistant acid phosphatase 'TRAP) membrane samples. After 1 or 14 days of culture, cells staining positive for TRAP were quantitated using a commercial staining kit Generation of osteoclasts from rheumatoid tissues Sigma). The population of cells digested from RA tissues that were adherent to glass coverslips contained many cells Identi®cation of resorption pit formation with the appearance of osteoclasts after culture for 24 h To assess the extent of bone resorption by cells in the Fig. 3a). These osteoclast-like cells were large and cocultures, dentine discs were examined for resorption TRAP-positive and contained many nuclei. This sug- lacunae on day 14, as described previously w14x. gested that osteoclast-like cells were resident in the 626 D. R. Haynes et al.

FIG. 2. PCR products corresponding to RANKL, OPG, RANK, M-CSF, TRAIL, TRAP and CTR mRNA detected by semi- quantitative RT-PCR for the 13 samples of RA tissue described in Table 1. Expression is shown relative to the housekeeping gene GAPDH. arthritic tissues. Many more osteoclastic cells were in the mean numbers of resorption pits and cells seen after 14 days of culture, at which time large expressing TRAP compared with cells incubated alone. multinucleated TRAP-staining cells were seen regularly Although OPG treatment resulted in complete inhibi- amongst cells cultured either alone Fig. 3b) or in tion of pit formation, there was no reduction in the the presence of human -like cells. Addition number of cells expressing TRAP, as stated above. of exogenous OPG did not markedly affect the number There is growing experimental evidence w7x that of TRAP-positive cells that formed from RA cells alone i) RANKL and OPG mRNA levels are good surrogates Fig. 3c) or RA cells cultured with human bone-derived for the expression of the corresponding , and cells. When RA cells were cultured alone on dentine ii) that osteoclastic resorption is dependent upon slices, large numbers of resorption lacunae were usually the effective concentration of RANKL, which is in seen by day 14 Fig. 3d). Slightly more resorption pits turn determined by the local concentration of OPG. were observed when RA cells were cultured with human We therefore compared the ratio of RANKL mRNA to bone-derived cells Fig. 3e). Treatment with 50 nguml OPG mRNA, as measured by RT-PCR, with the OPG completely inhibited the formation of resorption numbers of resorption pits produced by cells digested pits in cultures of RA cells alone Fig. 3f ) and in from the RA tissues cultured alone Fig. 4). There was cocultures of RA cells with human bone-derived cells. a strong positive correlation between these values Using inverted-phase microscopy, resorption pits could wSpearman non-parametric) correlation 0.762; be observed during the culture period, and no pits were P = 0.028x, consistent with a role for RANKL in pro- seen before day 7. The numbers of resorption pits moting osteoclastic bone resorption in human RA and TRAP-positive cells seen at day 14 are compared tissues. A similar comparison of the numbers of resorp- in Table 2. Culturing the RA cells in the presence tion pits with the ratio of TRAIL mRNA to OPG of human bone-derived cells resulted in a slight increase mRNA was carried out but no signi®cant correlation Factors regulating bone loss in RA joints 627

FIG. 3. a) Multinucleated cells expressing TRAP dark-staining cells) were present in the RA tissues and were seen after 24 h of culture of RA cells alone. Magni®cation 3250. b) Many more multinucleated cells expressing TRAP were seen after 14 days, when RA cells were cultured alone. Magni®cation 350. c) Treatment of these cells with 50 nguml OPG did not markedly reduce the numbers of cells expressing TRAP at day 14. Magni®cation 350. d) RA cells usually formed many resorption pits in dentine slices after 14 days when cultured alone. Magni®cation 3100. e) Slightly more resorption pits were seen when the RA cells were cultured with human bone-derived cells. Magni®cation 3100. f ) Treatment with 50 nguml OPG for 14 days completely inhibited the formation of resorption pits in cultures of RA cells alone. Magni®cation 3100. was noted P > 0.05), indicating that TRAIL may have rheumatoid cells to form resorption lacunae Table 2). less of a role than OPG and RANKL in regulating This inhibition occurred in the presence or absence osteoclast formation in this pathology. of added human bone cells. Interestingly, the numbers of cells expressing TRAP was only slightly reduced by Inhibition of osteoclast formation by OPG OPG treatment. Cells isolated from RA tissues were cultured on dentine slices in the presence of 50 nguml OPG to determine Discussion whether RANKL expressed by the cells was essential for the formation of osteoclasts. As shown pictorially This study has shown that bone-resorbing osteoclasts in Fig. 3, OPG completely inhibited the ability of can be generated in culture from cells present within the 628 D. R. Haynes et al.

TABLE 2. TRAP expression and resorption pit formation during synovial tissues. It is signi®cant to note the relationship 14 days of culture of cells isolated from RA tissue; the effects of between the ratio of RANKL mRNA to OPG mRNA coculture with human bone-derived cells NHB) and treatment with 50 nguml OPG are shown and osteoclast formation. These data suggest a correla- tion between the mRNA levels of RANKL and OPG +OPG and the corresponding levels. In addition, our ®ndings support the concept that the relative levels of RA cells RA cells RA cells RA cells RANKL and OPG is a key factor in determining bone +NHB alone +NHB alone loss in these tissues. Pits per dentine slice 88.8 " 28.0a 45.1 " 18.1 0.0 " 0.0 0.0 " 0.0 Although we found abundant expression of M-CSF TRAP+ cells per 106 287 " 56 196 " 50 223 " 41 176 " 35 mRNA in cells digested from RA tissues, it is clear that RA cells M-CSF, an essential cofactor for the induction of aMean " S.E.M. for n=8. osteoclast differentiation by RANKL, is limiting in human osteoclastogenesis in culture w4x. As in other studies w8, 13x, M-CSF was included in all our cell cultures as it allowed us to investigate the activities of RANKL independently of a requirement for M-CSF. The number of multinucleated TRAP-positive cells that developed from cells isolated from the rheumatoid tissues was not markedly reduced by OPG treatment, while OPG treatment totally prevented resorption pit formation, as reported previously w13x. This may indi- cate that osteoclast precursors in the rheumatoid tissues have differentiated further towards becoming mature osteoclasts than the less differentiated cells in the peri- pheral monocyte population, as TRAP expression is markedly inhibited by OPG in culture systems using peripheral blood monocytes w3, 4x. While development of the rheumatoid cells into multinucleated TRAP- positive cells may not be affected by inhibiting RANKL with OPG, RANKL does appear to be required to FIG. 4. Association of the ratio of RANKL mRNA to OPG stimulate and maintain osteoclastic bone resorption in mRNA with the number of resorption pits produced by cells these tissues. This concept is further supported by the digested from the same tissue samples. The data points were derived from the experiments described in Fig. 2 and Table 2. observation that TRAP mRNA was expressed by cells The individual samples are numbered as described in Table 1. isolated from most of the patient tissue samples, The regression analysis was carried out using the SAS version but strong expression did not always correlate with the 6.21 software package, and signi®cance was determined from formation of resorption pits by cells isolated from these the resulting r value. tissues. CTR expression may be a more appropriate indicator RA joint. The data indicate that these tissues contain of osteoclast differentiation than TRAP expression in both mature osteoclasts and their precursors, as well as this pathology. CTR was expressed in all the pannus producing factor s) essential for osteoclast formation. samples but in only one of the synovial membrane Our results show that RANKL mRNA is expressed in samples. This is consistent with a recent report, using cells within the RA tissues, and the ratio of RANKL in situ hybridization, showing that CTR mRNA is not mRNA to the mRNA of its inhibitor, OPG, expressed expressed in the synovial membrane but is expressed in the RA tissues signi®cantly correlated with the in the tissue±bone interface in RA w11x. CTR was also formation of functional osteoclasts. These results are consistently expressed in rheumatoid tissues from supported by recent reports showing the expression which osteoclasts readily formed, further supporting of RANKL and RANK in rheumatoid tissues in the concept that CTR expression is associated with the humans w11, 13x and in animal models w12, 19x. later stages of osteoclast differentiation. Our results differ from a previous report w8x which RANKL has been shown to be required for osteoclast found that additional rat osteoblast-like cells were formation, and is normally provided by osteoblast-like required for RA synovial macrophage and blood mono- cells in bone w3, 4x. Recent reports indicate that stromal cytes to produce osteoclastic resorption. The reason support may be provided by either synovial ®broblasts for the difference may be that we mainly used cells w11, 13x or lymphocytes present in the rheumatoid tissues isolated from tissue adjacent to bone, corresponding to w10, 12x. Horwood et al. w10x showed that CD3 + T cells the invading pannus, whereas the previous study used from the human rheumatoid joint express RANKL and synovial and blood monocytes from the can promote osteoclast formation from rodent RA patients. More studies would need to be carried out cell precursors. Kong et al. w12x demonstrated that, in to determine if indeed there are functional differences addition to the production of RANKL by lymphocytes, in osteoclast formation in these different regions of the inhibition of RANKL by OPG treatment in vivo Factors regulating bone loss in RA joints 629 reduced both bone and cartilage destruction in a model helpful advice. This work was supported by the of adjuvant arthritis in rats. Our study strongly supports National Health and Medical Research Council of these ®ndings and suggests that OPG may be similarly Australia and the University of Adelaide Faculty of ef®cacious in human RA. Medicine. Other mediators known to be produced by in¯am- matory cells in the soft tissues of the RA joint may promote bone loss indirectly by inducing the expression of RANKL. The in¯ammatory mediators References IL) -1b and TNF-a have been shown to stimulate 1. Gravellese EM, Harada Y, Wang JT, Gorn AH, Thornhill RANKL mRNA expression w20x, and TS, Goldring SR. 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